Role of the Zn₁and Zn₂sites in Metallo-β-lactamase L1

Abstract

In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogs of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn 1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)-and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn 2 site in L1. Heterobimetallic analogs (ZnCo and ZnFe) analogs of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogs were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is five-coordinate in the resting state, proceeds through a four-coordinate species during the reaction, and is five-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn 1 site is essential for catalysis in L1 and that the metal ion in the Zn 2 site is crucial for stabilization of the nitrocefinderived reaction intermediate.

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…Benkovic and co-workers (16,17) reported for the first time the accumulation of a reaction intermediate in the hydrolysis of nitrocefin by the B1 enzyme CcrA from B. fragilis. The same intermediate was also reported for the B3 enzyme L1 (37,46) and for an evolved mutant of the B1 lactamase BcII (39). In all cases, this intermediate was stabilized exclusively by the dinuclear forms of these enzymes.…”

“…The present spectrum shows a larger signal dispersion than that of mononuclear Co(II)-L1 (37). Kinetic parameters were derived from nonlinear fit of the Michaelis-Menten equation to initial rate measurements.…”

“…Here we report a series of experiments that allow us to propose that the metal site in GOB is involved in stabilization of an anionic intermediate that accumulates during catalysis, thus favoring C-N bond cleavage after the nucleophilic attack. These results are unprecedented because these intermediates have been only identified in catalysis by dinuclear lactamases (16,17,37,39) and disclose the existence of a common catalytic feature in M␤Ls from all subclasses.…”

“…These findings are puzzling because, although it is accepted that B2 lactamases may work without a metal-activated nucleophile (24,36), this is not the case for B3 lactamases (37). A crystal structure of mono-Zn(II) L1 has revealed that the single metal ion is located in the 3H site (i.e.…”

“…the one involved in nucleophile activation) (38). A recent kinetic and spectroscopic study in L1 has also indicated that mono-Zn(II) L1 in solution is active with the metal ion localized in the 3H site (37). Thus, there is no precedent of a B3 lactamase without a metal-activated nucleophile.…”